IMPC- Understanding somatic MSI+ colorectal cancers using conditional coding and non-coding mutations in Mlh1.

Lead Research Organisation: University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics

Abstract

Colorectal cancer affects 1 in 20 people in the western world. Mutations that cause colon cancer can be inherited or, more often, acquired during a patient's lifetime. One commonly mutated gene is MLH1 which is important for repairing mistakes or "mismatches" in DNA sequences. Mutation in the coding sequence of the MLH1 gene can be inherited, causing the famililal cancer syndrome called Lynch. However they are very rarely acquired in somatic tissues. Instead, MLH1 is normally repressed by chemical marks on the DNA (called DNA methylation or epimutation). Both inherited and acquired mutations in MLH1 disrupt DNA repair and lead to high levels of mutations in the DNA which are key to the development of cancer. However, the cancers that arise from the two types of mutations have important differences in their prognosis and response to treatment, so we need to investigate them using separate and appropriate systems.
Mouse models of cancer provide powerful ways of studying the underlying biology of cancer development because, unlike isolated cell line systems, they allow us to study tumour cells in an appropriate context or environment which can dramatically change the way they behave. Crucially they can also be used for the testing of potential treatments. Currently there are models carrying Mlh1 mutations that are representative of Lynch syndrome but none that "acquire" Mlh1 repression together with an appropriate combination of other mutations in colon cancer genes. In this project we will aim to do this using mice carrying a "conditional" mutation in Mlh1 that can be activated in specific tissues at specific times. We will combine the conditional mutation with a common cancer causing mutation in the Braf oncogene and activate them in cells within the intestine in adult mice. We will also combine a promoter mutation, similar to an epimutation, that partially represses Mlh1 expression with the same intestinal Braf mutation. We will then characterise the type, severity and biology of the cancers arising in mice carrying these mutation combinations. We propose that one or both of these models will provide a suitable system for testing existing and new drugs, alone or in combinations, specifically designed to treat the highly mutated cancers caused by acquired mutations in Mlh1.

Technical Summary

Microsatellite-unstable (MSI+) tumours are commonly found in the colon where they comprise ~15% of all cancers. MSI+ cancers can occur as a result of the familial Lynch syndrome which is caused by germline mutations in components of the mismatch repair pathway such as MSH2 and MLH1. MSI can also arise in sporadic cancers where, in the majority of cases, MLH1 is silenced by promoter methylation. Colorectal tumours arising in Lynch syndrome patients usually develop via the classical adenoma to carcinoma pathway. In contrast, sporadic MSI+ cancers develop from sessile serrated adenomas. This pathway is characterised by the presence of BrafV600E mutations and has a lower frequency of APC mutations than the classical pathway.
Sporadic MSI+ cancers with BrafV600E mutations and promoter silencing of MLH1 form a significant subgroup of colorectal cancer with a good prognosis but a differential response to common chemotherapies. A greater understanding of this group of cancers is essential to develop subtype specific or targeted treatments.
Germline Mlh1 mouse knockouts with and without Apc mutations have been studied and proved representative of Lynch syndrome but to date, no model of somatic Mlh1 coding mutation combined with BrafV600E mutation exists. In this proposal we aim to generate and characterise such a model using the IMPC Mlh1tm1c strain and compare it with an existing Mlh1 promoter mutation created in our laboratory. We will use these systems to investigate the initiation and progression of sporadic MSI+ tumours. Our long term goal will be to use either or both models to test existing and novel therapies, including immunological and epigenetic based approaches, in this distinct class of colorectal cancer.

Publications

10 25 50
 
Title The creation of a mouse model of mismatch repair deficient colorectal cancer 
Description We have generated a mouse model of mismatch repair deficient colorectal cancer by breeding mice containing a conditional BRAFV600E oncogenic mutation with MLH1 conditional knockout mice. These mutations have been activated specifically in the intestine and result in increased numbers of polyps and faster growth than those found in mice carrying the BRAFV600E mutation alone. Preliminary analysis shows that these polyps are have microsatellite instability. We need to carry out further analysis but it is likely that this model will prove valuable for investigating the effects of exisiting and novel therapeutic technologies on intestinal cancers with microsatellite instablity. We are particularly interested in the mechanism by which immunotherapy successfully treats these cancers. Our collaborators, Dr David Church and his group are generating further MLH1 mouse crosses with APC mutated and POLE mutated strains. They are also developing methods to assess and compare immune activity in tumours from all three crosses. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? No  
Impact We are still working on our analysis of this model and a comparison with other similar mouse strains. We expect the model to be useful for investigating the effects of exisiting and novel therapies on mismatch repair deficient colorectal cancer. 
 
Title Using next generation sequencing to detect allele specific biases 
Description We have used Illumina Miseq technology to accurately determine allele-specific differences in gene expression, DNA methylation and protein binding. This method is sensitive and reproducible, and can be used in cell line and animal models and patient samples. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact This has enabled us to measure the effect rs1800734 genotype on intermediate cancer phenotypes to uncover the mechanism by which it predisposes to colorectal cancer. 
 
Description David Church and laboratory 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated a mouse model of microsatellite unstable colorectal cancer by breeding mice containing a conditional BRAFV600E oncogenic mutation with MLH1 conditional knockout mice. These mutations have been activated specifically in the intestine and result in increased numbers of polyps and faster growth than those found in mice carrying the BRAFV600E mutation alone. Preliminary analysis shows that these polyps are have microsatellite instability.
Collaborator Contribution Dr Church and his group are generating further MLH1 mouse crosses with APC mutated and POLE mutated strains. They are also developing methods to assess and compare immune activity in tumours from all three crosses.
Impact A new mouse model of MSI+ colorectal cancer with the potential for two further models of hypermutated colorectal cancer. This project is very much a work in progress at the moment with encouraging preliminary results.
Start Year 2019
 
Description Speaker at Alumni symposium at St Catharine's College Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The symposium was intended to bring together researchers from a wide range of disciplines to discuss the nature and impact of their work with a non-specialist student audience. The main aim was to inform and encourage discussion with the audience.
Year(s) Of Engagement Activity 2018
URL https://mcr.caths.cam.ac.uk/symposium